RNA transcripts of cloned, synthesized, or isolated DNA segments are useful in studying RNA splicing, sequencing DNA segments, and in vitro translation. RNA transcription may be used for RNA sequencing, in producing proteins, and for post-translational modification of proteins. Also, RNA translation may be used to produce more stable bio-pharmaceuticals such as DNA/RNA chimeras.
In vitro transcription of DNA via RNA polymerases has been used as a step in producing mRNA. The mRNA is in turn used to produce functionally active proteins. Such production of mRNA has been proposed as a means for large scale industrial manufacture of polypeptides. It would provide the milligram or more quantities of mRNA required for large industrial processes.
Bacteriophage RNA polymerases are highly active and preferred for in vitro transcription of DNA. They are composed of a single polypeptide chain, while other RNA polymerases tend to be more complex or have one or more co-factors. The T7, T3, and SP6 RNA polymerases have all been cloned. They are commercially available in large quantities, and can also be readily produced from commerically available vectors.
The RNA polymerase-specific DNA promoter sequences found at the beginning of most genes define the high affinity RNA polymerase binding site from which transcription initiation proceeds. The promoter sequence of a particular RNA polymerase is a DNA segment attached to the template DNA strand upstream from the portion to-be-transcribed into RNA (i,e., at the 5'-end of the template). The promoter allows the RNA polymerase to attach to the DNA template sequence near the site to-be-transcribed, so that transcription may be initiated.
It is generally regarded that each species of RNA polymerase is specific for a particular promoter sequence. Each of the three bacteriophage RNA polymerases (T7, T3, and SP6) has its own specific DNA promoter sequence.
After the RNA polymerase has attached to the promoter sequence of the template and transcription has been initiated, the promoter sequence releases the RNA polymerase. The RNA polymerase then continues transcription along the template until (1) it runs out of RNA starting materials, (2) the transcription reaction is terminated by a particular nucleotide, nucleoside or analog, (3) transcription is spontaneously aborted when the RNA polymerase and the template prematurely disassociate, or (4) the end of the DNA template is reached.
Most DNA-dependant RNA polymerases have been shown to accept RNA as a template for polymerization of NTPs. Such polymerization has been characterized as usually being inefficient and nonspecific for both transcription initiation and termination. The DNA-dependant polymerase of T7 will accurately and efficiently replicate a short RNA template under some circumstances. See, Konarska et al., Cell Vol. 57, 423-431 (1989). The short single-stranded RNA template does not have appropriate promoter sequences. Rather, it has a specific binding site to which the T7 RNA polymerase attaches and begins transcription. This was discovered by Konarska et al after high levels of unanticipated RNA side-products (called X-RNA or Y-RNA) were generated without any explanation. Other unexplained side products observed were head-to-tail RNA polymers and complementary double-stranded RNA of the X-RNA or Y-RNA.
Additional experiments by Konarska et al., Cell Vol. 63, 609-618 (1990) showed that the X-RNA or Y-RNA served as a template for itself. The origin of X-RNA or Y-RNA was unclear. The only sources for the X-RNA and Y-RNA have been some preparations of T7 RNA polymerase. Contamination of the preparations with X-RNA, Y-RNA, or a precursor was stated as the likely reason for the RNA side products.
Obtaining RNA products from these X- or Y-RNA templates was puzzling since the templates did not have the DNA promoter sequence specific for the attachment of the T7 RNA polymerase. Konarska et al. also conducted experiments showing that other RNA polymerases did not produce RNA products from X- or Y- RNA templates unless a promoter was attached.
The Konarska et al. products contained palindromic sequences (tandem inverted repeats), which are known to be recognition sites for some RNA polymerases. Thus, certain segments along these RNA templates (possibly a palindromic sequence) were able to specifically bind the T7RNA polymerase tightly enough to permit transcription of the RNA template to begin. None of the naturally occurring RNAs tested (including total RNA from E. coli or Hela cells and tRNA from E. coli, yeast, or Hela tRNA) served as an efficient template for transcription by T7 RNA polymerase. Moreover, it was noted that when these other RNAs were present, replication of X-RNA was strongly inhibited.
Thus, Konarska et al. state that replication of the X-RNA or Y-RNA was specific to T7 RNA polymerase. The very closely related T3 RNA polymerase (80% homology with T7 RNA polymerase) was unable to replicate the X-RNA or Y-RNA without a promoter sequence.
Lewis et al. (J. Biol. Chem., 255, No. 10, 4928-4936 (1980)) showed that wheat germ RNA polymerase II will transcribe one of the nicked strands of a double-stranded Simian Virus 40 DNA to produce a complementary RNA strand without a DNA promoter. Wheat germ RNA polymerase II has a large, complex structure, which is similar in structure and sensitivity to mammalian RNA polymerase II. One of the two DNA strands must be nicked to provide a 3'-hydroxy end for transcription to begin, since no transcription was observed with intact DNA. Lewis et al. reported that DNA transcription with RNA polymerases is neither specific or very controllable. A DNA strand must be nicked to start transcription, which ends when the RNA polymerase encounters another nick in the double-stranded DNA.
Moreover, Lewis et al. reported that transcription of deproteinized DNA by wheat germ polymerase II in vitro was doubtful. Specifically, a DNA-binding protein co-factor was thought necessary to effect biologically relevant transcription. Apparently, the binding protein co-factor serves as a bridge which binds the RNA polymerase and the DNA template until translation of the template is progressing. Thus, the binding cofactor protein replaces the binding provided by a promoter sequence specific for the RNA polymerase.
Production of RNA by wheat germ RNA polymerase from nicked double-stranded DNA proceeds slowly. As transcription occurs, the double strands of DNA must be melted (made to unwind) to provide a single DNA template strand for translation. Some of the products are hybrid DNA/RNA strand (chimeras) and some of the products are entirely RNA, but there is apparently limited control over which products will be obtained. The nicking process is unpredictable as to the location of nicks along an uncharacterized DNA sequence, and the subsequent RNA products are also unpredictable.
E. coli RNA polymerase, in the presence of its binding co-factor protein (DNA binding protein I), can transcribe a DNA template to produce a RNA chain. (See, Geider et al., Proc. Natl. Acad. Sci., U.S.A., 75, No. 2, 645-649 (1978)). No nicking is required, and there is apparently limited control over which products will be obtained. The transcription is random as to the initiation location along a DNA sequence, and the subsequent RNA products are also unpredictable. (See also, Lewis et al., cited above).
The bacteriophage T7 RNA polymerase, as is typical for other RNA polymerases, is very specific for its particular promoter sequence. It is a single sub-unit polypeptide that can transcribe genes in vitro from highly conserved T7 promoter sequences in the absence of other proteins (Chamberlain et al., The Enzymes, Boyer, ed. New York Acad. Press, 3rd ed., p. 85 (1982); Dunn et al., M. Mol. Biol. 166, 477-535 (1983)). The efficiency and high promoter specificity of T7 RNA polymerase has made it useful for in vitro generation of small mRNA (Geider, Proc. Natl. Acad, Sci. USA 75, 645-649 (1978)) as well as for cloning and in vivo gene expression (Guruvich et al., Analytical Biochem 195, 207-213 (1991); Lewis et al., J. Biol. Chem. 255, 4928-4936 (1980); Milligan et al., Methods Enzymol. Vol. 180a, ed., 50-52 (1989)). Because of its simplicity and minimal requirements T7 RNA polymerase has been practical for studying the complex mechanisms involved in transcription.
However, there is a significant difficulty encountered with the use of T7 RNA polymerase for generating RNA transcripts. Large quantities of contaminating short RNA molecules in the range of 2-8 NTPs are generated, which comprise incomplete RNA translation products. These short chains are believed to be due to premature release of the RNA polymerase by the promoter sequence. The RNA polymerase apparently discontinues translating the template after its release by the promoter.
As a result of the large numbers of abortive initiation side products, the yield of the desired transcript from T7 mediated transcription is low. The abortive side products typically result in a yield which is an order of magnitude lower than the yield expected from the NTP reagents consumed. This has required use of terminator NTPs as marking units, and separation of short RNA molecule contaminants from marked terminated sequences as part of RNA sequencing procedures.
Further, when double stranded DNA is used as the template for T7 transcription, the RNA polymerase must melt an eight base pair DNA segment. This allows for base pair complementarity between the eight base pair segment and the template strand. The melting must continue as the double strand is unwound and transcribed.
Accordingly, there is a need for efficiently generating RNA transcripts without producing large amounts of short RNA molecules as contaminating side-reaction products. Particularly, there is a need to efficiently produce long chain RNA transcripts. Also, there is a need for a method to transcribe a DNA template or RNA template without requiring either an RNA polymerase-specific DNA promoter sequence or an RNA polymerase-specific binding co-factor protein. Further, there is a need for a method of transcribing a DNA template, which is not part of two substantially complementary strands that have been nicked to provide RNA transcription initiation and termination sites.